This project concerns signaling mechanisms in cells of the reproductive axis. Electrical excitability and intracellular signaling will be studied in male mouse germ-line cells and epididymal spermatozoa and in male rat pituitary gonadotropes. The physiology of single living cells will be studied by patch clamp and optical indicators. The guiding hypotheses are that membrane potential, intracellular Ca2+, and cyclic AMP are key cellular signals that control steps in reproduction. The ion channels of spermatids and sperm will be identified by patch clamp and immunocytochemistry, with special attention to voltage-gated Ca2+ channels. In sperm and spermatids the actions of bicarbonate on cyclic AMP, protein phosphorylation, motility, and ion channel function will be compared. Mechanisms regulating Ca2+ clearance from sperm and spermatid cytoplasm will be identified. In gonadotropes, immunocytochemical experiments will identify the IP3 receptors present, and quantitative multicompartmental Ca2+ measurements will characterize mitochondria! uptake and buffering of Ca2+ to permit formulation and test of kinetic models for cellular Ca2+ dynamics during the Ca2+ oscillations induced by gonadotropin releasing hormone. These studies will identify potential targets for future male contraceptives.